β-ARs stimulated by pharmacological agonists in vivo (presumably in concert with circulating catecholamines) promote survival and ameliorate inflammation in rodent models of endotoxemia. However, the extent to which such findings are relevant to the native actions of β-ARs in the setting of endotoxemia/septic shock is unclear, since many of the β-AR agonists employed exhibit a relative lack of specificity, and likely differ from endogenous circulating catecholamines (whose levels are increased in response to LPS) in their temporal and spatial activity in vivo. We therefore extended these studies to evaluate the impact of β-ARs modulated by their native ligands, the catecholamines, on LPS-mediated inflammation and mortality. Specifically, we have utilized mice lacking β-AR function to demonstrate for the first time that β-AR stimulation by endogenously released catecholamines increases survival and reduces inflammation in the setting of endotoxemia.
A brief comparison of the results of the present endotoxemia study evaluating the role of endogenous catecholamines with those of previous studies evaluating the impact of pharmacological β-AR agonists, yields some interesting similarities and differences. Pre-treatment of LPS-challenged rodents with the β2-AR agonist, terbutaline, resulted in inhibition of TNF-α and augmentation of IL-10 production by similar magnitudes to those observed in our study, and also increased survival [16
]. Similarly, pre-treatment of LPS-challenged mice with the broad-spectrum β-AR agonist isoproterenol resulted in decreased TNF-α serum levels [15
]. In contrast to our study however, LPS-challenged rodents pre-treated with isoproterenol or the β2-AR-selective agonist clenbuterol exhibited reduced levels of serum IL-12p40 [30
] or IL-1β and IL-6 [17
] respectively. We observed that the LPS-induced serum levels of these three cytokines did not differ between β-less and wildtype mice. The differential cytokine responses observed may reflect important differences in the actions of endogenous catecholamines versus pharmacological agonists on β-AR function not just in terms of their receptor specificity, but also their relative activity or tissue accessibility. With respect to activity, the pharmacological agonists employed in these studies are presumably stimulating β-ARs beyond the level already achieved by endogenous catecholamines. One example of differential tissue accessibility is that catecholamines have access to β-AR expressed in the CNS, whereas the β-AR agonists are inefficient at crossing the blood-brain barrier. An alternative explanation is that disruption of β-AR gene function may have activated secondary changes during development that compensate for lack of β-AR regulation of these particular cytokines, but not TNF-α and IL-10. In summary, our data demonstrate that β-ARs respond to endogenous catecholamines released in response to LPS challenge by decreasing the inflammatory response and increasing survival.
Having established that β-ARs serve a protective role in endotoxemia in this genetic knockout model, we were then able to determine the extent to which β-ARs expressed on bone marrow-derived versus radioresistant cells contribute to survival and inflammation in the setting of endotoxemia. Analysis of reciprocal bone marrow chimeras revealed that β-ARs expressed on radioresistant cells played the predominant role in promoting survival, since the survival rate of β-less mice reconstituted with wt bone marrow was about as poor as that of mice reconstituted with β-less marrow. Nonetheless, β-ARs expressed on hematopoietic cells must also contribute to some extent to survival, since wt mice reconstituted with β-less marrow exhibited increased mortality relative to those reconstituted with wt marrow. The data suggest that β-ARs expressed on radioresistant cells play an essential role in protecting from LPS-induced death regardless of whether or not β-ARs are expressed on bone marrow-derived cells, whereas the protective role of β-ARs present on the latter compartment only becomes apparent when β-AR function on radioresistant cells is intact. In a subsequent set of experiments, we compared the cytokine responses of LPS-challenged bone marrow chimeras in order to determine whether β-AR location played an important role in inflammatory responses. We observed that the TNF-α response of mice lacking β-ARs only on radioresistant cells was significantly higher than that of mice that retained β-AR function on all cells, demonstrating that β-ARs expressed in this compartment not only play an important role in survival, but also in inhibition of LPS-induced TNF-α responses in vivo. Consistent with this observation, the TNF-α response of mice lacking β-ARs on radiosensitive hematopoietic cells alone was only marginally increased relative to (and not statistically significant from) those animals with wt cells. Although we did observe differences in the IL-10 responses of reciprocal chimeras compared to WW or BB controls, they did not reach statistical significance. For simplicity’s sake, the relative mortality exhibited by the 4 experimental groups can be represented as BB≥WB<<BW>WW, and the relative TNF-α response as BB≥WB<<BW≥WW. Thus, the relative order of the 4 groups is similar with respect to mortality and TNF-α responses. Although these data do not demonstrate a direct correlation between mortality and TNF-α levels, they do reveal a dominant role for β-AR expressed on radioresistant cells in promoting survival and decreasing pro-inflammatory TNF-α responses in vivo. Our data do not preclude a role for β-ARs expressed on immune cells to dampening of pro-inflammatory TNF-α responses, but suggest that it is subordinate to those expressed on radioresistant cells.
Increased survival may be due to β-AR actions that reduce TNF-α production per se
, and to those that modulate other protective mechanisms. With regard to the former, an attenuated TNF-α response could result in decreased expression of lethality mediators that are induced much later in the cytokine cascade. Recent studies have demonstrated that the major source of LPS-induced TNF-α in vivo
is the macrophage population, and mice with a macrophage-specific defect in TLR expression are relatively resistant to LPS-induced death [1
]. Mice reconstituted with bone marrow lacking TLR-4 exhibit a markedly decreased TNF-α response to LPS challenge, highlighting radiosensitive macrophages as a major source of this cytokine in the setting of endotoxemia [31
]. The TNF-α response of these chimeric mice is still readily detectable however, suggesting that radioresistant cells do contribute to this inflammatory response [31
]. Our observation that radioresistant cells play a significant role in β-AR-mediated inhibition of TNF-α responses is intriguing. One possible explanation is that this radioresistant population may comprise resident macrophages that fail to be efficiently replaced by donor cells in the transplant setting. For example, the lung and liver contain relatively radioresistant macrophage populations [32
]. An alternative explanation for the involvement of the radioresistant compartment in β-AR-modulation of TNF-α production is that β-ARs expressed in the CNS act in an indirect manner to suppress TNF-α production by radiosensitive macrophages in the periphery. Catecholamines are released within the CNS in response to systemic LPS administration, and β-ARs are expressed on microglia, astrocytes and neurons [35
]. Central β-AR stimulation has the potential to modulate peripheral immune responses via regulation of sympathetic tone and activation of the hypothalamic-pituitary-adrenal axis for example. Indeed, depletion of central catecholamines by chemical lesioning of dopaminergic neurons results in impaired peripheral host immunity [38
An alternative explanation for the protective role of β-ARs expressed on radioresistant cells is that they can directly protect against LPS-induced mediators of lethality. For example, β1-, β2-, and to a lesser extent β3-AR, are expressed on cardiac myocytes where they regulate cardiac contractile activity. Specifically, β1- and β2-ARs mediate the inotropic and chronotropic effects of catecholamines, whereas the effect of β3-AR is less clear. However, sepsis/endotoxemia is associated with a net decrease in cardiac contractile activity despite elevated sympathetic tone, and available evidence suggests that this is due, at least in part, to inhibition of the positive inotropic action of β1/β2-AR [19
]. It is therefore possible that the increased mortality of β-less relative to wt mice is due, at least in part, to markedly deficient cardiac contractile activity secondary to loss of positive inotropic β1/2-AR action on myocytes. The elevated TNF-α levels present in LPS-challenged mice lacking β-ARs could contribute to this effect. Similarly, lack of β-AR function on endothelial cells may increase the severity of microvessel hyperpermeability associated with mortality in the setting of endotoxemia. β-ARs expressed in the CNS may also play a role in promoting survival, by acting indirectly to promote cardiac output and protect against tissue injury. For example, systemic administration of LPS has been reported to stimulate the synthesis and/or projection of catecholamines in several areas of the CNS [35
], and activation of central β-AR has been reported to increase sympathetic outflow to the heart [40
In order to examine the role of β1- and β2-ARs per se
to the increased mortality and inflammation we observed in the β-less mice, we evaluated these parameters in mice with intact β3-AR function, but lacking functional β1- and β2-ARs. β3-ARs are primarily expressed in adipose tissue where they mediate lipolytic and thermogenic responses. β3-AR expression in myocardium is markedly elevated in the setting of sepsis [19
], but our current understanding of β3-AR function in inflammation or vascular/cardiac function is relatively limited. As observed for mice lacking all three β-AR subtypes, the double knockout mice exhibited increased mortality relative to wt controls. The relative increase in mortality between β1/2−/− and control mice approached that seen for the β-less study at 24 hours post-LPS injection. In addition, as observed for β-less mice, β1/β2−/− mice exhibited increased TNF-α and decreased IL-10 serum levels in response to LPS challenge relative to wt animals. Caution should be taken in making a direct comparison between the β-less and double knockout mice in this study, because the control mice in each case exhibited differential sensitivity to LPS, presumably because of differences in their respective genetic backgrounds. Nevertheless our data demonstrate that β3-ARs do not play a major role in promoting survival and decreasing inflammation in the setting of endotoxemia.
In summary, our data demonstrate that β1- and β2-ARs play the dominant protective role in the setting of endotoxemia, and respond to endogenous catecholamines by increasing survival and dampening inflammatory responses initiated by LPS. Although our data suggest that β-ARs expressed on immune cells can contribute, it appears that β1- and β2-ARs expressed on radioresistant cells play the major role in inhibiting LPS-induced lethality and TNF-α responses. It is likely that the pro-survival actions of β-AR are mediated both by dampening the acute TNF-α response to LPS and by promoting cardiac/vascular function. It will be of great interest to identify the radioresistant tissues whose β-AR function plays such a critical role in protecting against LPS-induced mortality.